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Objective Petrodentine, the core of the lungfish tooth plate, is a well-mineralized tissue similar to mammalian enamel and analogous to enameloid in fish teeth. Petrodentine is formed solely by petroblasts, which are specialized odontoblasts, whereas enameloid is a composite tissue produced by both odontoblasts and dental epithelial cells. To clarify the details of petrodentine formation, petroblasts were investigated using histochemical and immunohistochemical techniques. Methods Extant lungfish (Lepidosiren paradoxa) were used in this study. Tooth plates during the stage of petrodentine formation were observed by means of histochemistry and immunohistochemistry. Commercial kits were used to detect enzyme activity. Correlative sections were immunostained using antibodies against selected peptides. Routine staining such as periodic acid-Schiff (PAS) reaction to identify glycogen and Elastica van Gieson staining for the detection of elastic fibers in histological sections were performed. In addition, conventional transmission electron microscopy was used for observing the fine structure. Results Petroblasts showed marked acid and alkaline phosphatase activities, and positive immunoreactivities against anti-nestin, anti-V-ATPase, and anti-Ca2+-ATPase, during the maturation stage, but in the matrix formation stage, reactions were much weaker than that of the maturation stage. During the maturation stage, petroblasts showed intense PAS reactivity, and glycogen particles were observed in petroblasts by transmission electron microscopy. Glucose transporter 1-immunoreactivity was observed in petroblasts in the matrix formation stage and the initial to mid part of the maturation stage. Conclusions The results in this study suggested that petroblasts have two functional stages, matrix formation and maturation, and glycogen plays an important role in the modulation of petroblasts.

Enameloid is a well-mineralized tissue covering the tooth surface in fish and it corresponds to the outer-most layer of dentin. It was reported that both dental epithelial cells and odontoblasts are involved in the formation of enameloid. Nevertheless, the localization and timing of secretion of ectodermal enamel matrix proteins in enameloid are unclear. In the present study, the enameloid matrix during the stages of enameloid formation in spotted gar, Lepisosteus oculatus, an actinopterygian, was examined mainly by transmission electron microscopy-based immunohistochemistry using an anti-mammalian amelogenin antibody and antiserum. Positive immunoreactivity with the antibody and antiserum was found in enameloid from the surface to the dentin-enameloid junction just before the formation of crystallites. This immunoreactivity disappeared rapidly before the full appearance of crystallites in the enameloid during the stage of mineralization. Immunolabelling was usually found along the collagen fibrils but was not seen on the electron-dense fibrous structures, which were probably derived from matrix vesicles in the previous stage. In inner dental epithelial cells, the granules in the distal cytoplasm often showed positive immunoreactivity, suggesting that the enamel matrix protein-like proteins originated from inner dental epithelial cells. Enamel matrix protein-like proteins in the enameloid matrix might be common to the enamel matrix protein-like proteins previously reported in the collar enamel of teeth and ganoine of ganoid scales, because they exhibited marked immunoreactivity with the same anti-mammalian amelogenin antibodies. It is likely that enamel matrix protein-like proteins are involved in the formation of crystallites along collagen fibrils in enameloid.

Although most fish have no enamel layer in their teeth, those belonging to Lepisosteus (gars), an extant actinopterygian fish genus, do and so can be used to study amelogenesis. In order to examine the collar enamel matrix in gar teeth, we subjected gar teeth to light and electron microscopic immunohistochemical examinations using an antibody against bovine amelogenin (27 kDa) and antiserum against porcine amelogenin (25 kDa), as well as region-specific antibodies and antiserum against the C-terminus and middle region, and N-terminus of porcine amelogenin, respectively. The enamel matrix exhibited intense immunoreactivity to the anti-bovine amelogenin antibody and the anti-porcine amelogenin antiserum in addition to the C-terminal and middle region-specific antibodies, but not to the N-terminal-specific antiserum. These results suggest that the collar enamel matrix of gar teeth contains amelogenin-like proteins and that these proteins possess domains that closely resemble the C-terminal and middle regions of porcine amelogenin. Western blot analyses of the tooth germs of Lepisosteus were also performed. As a result, protein bands with molecular weights of 78 kDa and 65 kDa were clearly stained by the anti-bovine amelogenin antibody as well as the antiserum against porcine amelogenin and the middle-region-specific antibody. It is likely that the amelogenin-like proteins present in Lepisosteus do not correspond to the amelogenins found in mammals, although they do possess domains that are shared with mammalian amelogenins.

In order to compare its characteristics with those of jaw tooth collar enamel, normally developing and experimentally regenerating ganoine from ganoid scales of Lepisosteus oculatus (spotted gar), an actinopterygian fish species, was examined by Western blotting and immunohistochemistry. Amelogenin, a major enamel matrix protein (EMP), is widely found from sarcopterygian fish to mammals. Therefore, we used antimammalian amelogenin antibodies and antisera: an antibody against bovine amelogenin; antiserum against porcine amelogenin; and region-specific antibodies or antiserum against the C-terminus, middle region, or N-terminus of porcine amelogenin in this study. Positive immunoreactivity with the antibody against bovine amelogenin, antiserum against porcine amelogenin, and the middle and C-terminal region-specific antibodies was detected in both normally developing and regenerating ganoine matrix, as well as in granules found within inner ganoine epithelial cells. These immunohistochemical analyses indicated that the Lepisosteus ganoine matrix contains EMP-like proteins with epitopes similar to mammalian amelogenins. In Western blotting analyses of regenerating ganoid scales with the antibovine amelogenin antibody, two protein bands with molecular weights of approximately 78 and 65 kDa were detected, which were similar to those found in Lepisosteus tooth enamel. Our study suggests that in Lepisosteus, EMP-like proteins in the ganoine matrix corresponded to those in tooth enamel. However, it was revealed that the 78 and 65 kDa EMP-like proteins were different from 27 kDa bovine amelogenin.

This is the first report on the stratum intermedium in vertebrates other than mammals. The aim of this study is to elucidate the fine structure and cytochemical features of the stratum intermedium during the stages of enameloid formation in Lepisosteus. Inner dental epithelium, stratum intermedium, stellate reticulum, and outer dental epithelium are consistently present in the tooth germs of Lepisosteus. The stratum intermedium cells are oval in shape, contain elliptical nuclei, and extend many small processes. It is implied that the structure of the enamel organ is different among actinopterygians, and that constitution of the enamel organ in Lepisosteus resembles that in higher vertebrates. Marked Ca-ATPase activity is observed at the cell membrane of the stratum intermedium cells, suggesting that the cells are involved in calcium transport during the stages of enameloid formation.

Ultrastructural and cytochemical studies on the mineralization of enameloid were performed using Heterodontus japonicus, an elasmobranch, and Tilapia buttikoferi, a teleost as materials. The mineralization of the enameloid in the Heterodontus was divided into the following two steps: (1) initial crystallization in the tubular vesicles that originated from the odontoblasts, and (2) crystal growth that was accompanied by the degeneration and removal of the organic matrix around the crystals. In the Tilapia, the mineralization of the cap enameloid followed three steps: (1) initial crystallization at the matrix vesicles, (2) aggregation of fine slender crystals along collagen fibrils, and (3) crystal growth with the degeneration and removal of the organic matrix. The pattern of early mineralization and the composition of organic matrix in enameloid were considerably different between the two species examined, while in both species the odontoblasts were mainly involved in the formation of the organic matrix of enameloid and in the initial mineralization. In the next step, remarkable crystal growth associated with the degeneration and removal of the organic matrix occurred in both the elasmobranch and the teleost species. The absorptive functions of the dental epithelial cells in the later stages of enameloid formation is probably similar in the two types of enameloid, and is essential for the production of well-mineralized enameloid.

The dental epithelial cells and enameloid at the stages of enameloid matrix formation, mineralization and maturation in the teleosts Oreochromis niloticus and Tilapia buttikoferi were investigated by means of enzyme histochemistry in order to identify their functions associated with the structural modification. No marked enzyme activities were found in the inner dental epithelial cells in the stage of enameloid matrix formation, although the outer dental epithelial cells often exhibited moderate alkaline phosphatase (ALPase) activity. In the stages of enameloid mineralization and maturation, the inner dental epithelial cells, which possessed a ruffled border at the distal ends, showed intense ALPase activity at their lateral and proximal cell membranes. At the same time, many acid phosphatase (ACPase)-positive vesicles and granules were localized at the distal cytoplasm of the inner dental epithelial cells. The outer dental epithelial cells, which contained well-developed labyrinthine canalicular spaces, showed neither marked ALPase nor ACPase activity. It is postulated that the dental epithelial cells in these two teleosts are mainly involved in the removal of the organic matrix from the enameloid, and in material transport to the enameloid during the later half of odontogenesis.

Tooth germs were examined by light and transmission electron microscopy. Collagen fibrils were relatively dispersed and thin at the early and middle stages of formation of the enameloid matrix, when the enameloid layer was thin. At the late stage, the fibrils became thicker, reaching nearly 30 nm dia, and formed the interwoven thick bundles that are characteristic of teleost cap enameloid. Abundant flocculent and/or fine, network-like material, probably representing glycosaminoglycans or proteoglycans, was located between the collagen fibrils. Tall, columnar, inner dental epithelial cells contained abundant rough endoplasmic reticulum and many mitochondria, and a well-developed Golgi apparatus was seen around the nuclei at the late stage. Elongated vesicles enclosing fine, filamentous material that resembled procollagen granules, and large granules containing fibril-like structures that were 150 nm in thickness and had periodic cross-banding at 32-nm intervals, were usually observed near the Golgi apparatus. The contents of the large granules were well stained with phosphotungstic acid, which suggests that inner dental epithelial cells synthesize collagen fibrils. At this time, odontoblasts also contained abundant rough endoplasmic reticulum and mitochondria, a well-developed Golgi, several kinds of granule including those that probably contained procollagen, and many microtubules. It is proposed that odontoblasts are involved in the formation of a considerable portion of the enameloid matrix, including collagen fibrils.

This is the first detailed report about the collar enamel of the teeth of Polypterus senegalus. We have examined the fine structure of the collar enamel and enamel organ of Polypterus during amelogenesis by light and transmission electron microscopy. An immunohistochemical analysis with an antibody against bovine amelogenin, an antiserum against porcine amelogenin and region-specific antibodies or antiserum against the C-terminus, middle region and N-terminus of porcine amelogenin has also been performed to examine the collar enamel matrix present in these teeth. Their ameloblasts contain fully developed Golgi apparatus, rough endoplasmic reticulum and secretory granules. During collar enamel formation, an amorphous fine enamel matrix containing no collagen fibrils is found between the dentin and ameloblast layers. In non-demineralized sections, the collar enamel (500 nm to 1 μm thick) is distinguishable from dentin, because of its higher density and differences in the arrangement of its crystals. The fine structural features of collar enamel in Polypterus are similar to those of tooth enamel in Lepisosteus (gars), coelacanths, lungfish and amphibians. The enamel matrix shows intense immunoreactivity to the antibody and antiserum against mammalian amelogenins and to the middleregion- and C-terminal-specific anti-amelogenin antibodies. These findings suggest that the proteins in the enamel of Polypterus contain domains that closely resemble those of bovine and porcine amelogenins. The enamel matrix, which exhibits positive immunoreactivity to mammalian amelogenins, extends to the cap enameloid surface, implying that amelogenin-like proteins are secreted by ameloblasts as a thin matrix layer that covers the cap enameloid after enameloid maturation.

Crystals and the organic matrix in both the enameloid and the maturation stage in this teleost fish were viewed by scanning and transmission electron microscopy. For transmission electron microscopy, specimens were demineralized by basic chromium (III) sulphate or phosphotungstic acid. Large numbers of defined compartments, thought to represent the outline of enameloid crystals, were seen in the outer layer of mature cap enameloid after this demineralization. However, the compartments were both less numerous and less well defined in the inner layer. These compartments suggest the existence of an organic enameloid matrix, which may correspond to the enamelins in mammalian enamel. As these defined compartments occurred in the areas of the tooth where large enameloid crystals were present, it is possible that the organic matrix constituting these compartments is involved in the production of large crystals.

Tooth germs during cap enameloid formation stages in Polypterus senegalus were investigated by transmission electron microscopy and enzyme histo- and cytochemistry. Enameloid formation was divided into three stages: matrix formation, mineralization, and maturation. The enamel organ consisted of the inner dental epithelial cells, stellate reticulum, and outer dental epithelial cells during cap enameloid formation stages, but no stratum intermedium was found. During the matrix formation stage, the tall inner dental epithelial cells contained well-developed Golgi apparatus, abundant cisternae of rough endoplasmic reticulum and mitochondria. Spindle-shaped vesicles containing a filamentous structure were seen in the distal cytoplasm. During mineralization and maturation stages, many ACPase positive lysosomes were present in the cytoplasm, whereas the organelles were decreased in number. The infoldings of the distal plasma membrane of the inner dental epithelial cells were visible in the mineralization stage but were not marked in the maturation stage. The activity of nonspecific ALPase, Ca-ATPase, and K-NPPase was detected at the plasma membrane of the inner dental epithelial cells during the stages of mineralization and maturation. The results of fine structure and enzyme cytochemistry suggested that the dental epithelial cells were mainly involved in the degeneration and removal of enameloid matrix and in material transportation during the enameloid mineralization and maturation stages, rather than in the enameloid matrix formation. The results also showed that the structure of the dental epithelial cells in Polypterus was different from that in teleosts and gars, but that the function of the dental epithelial cells was similar to that in teleosts possessing well-mineralized cap enameloid.

During cap enameloid formation in gars (Lepisosteus oculatus), the dental epithelial cells that constitute the enamel organ were observed by means of transmission electron microscopy and enzyme cytochemistry to detect the hydrolytic enzyme activities, alkaline phosphatase (ALPase), acid phosphatase (ACPase), calcium-dependent adenosine triphosphatase (Ca-ATPase) and potassium-dependent p-nitrophenylphosphatase (K-NPPase) (sodium, potassium-activated adenoshine triphosphatase (Na-K-ATPase)). The enameloid formation process in gars was divided into three stages: matrix formation, mineralisation and maturation. The enamel organ consisted of the outer dental epithelial (ODE) cells, stellate reticulum (SR), stratum intermedium (SI) and the inner dental epithelial (IDE) cells during the whole of the cap enameloid formation stages. During the matrix formation stage, many cisternae of rough endoplasmic reticulum and widely distributed Golgi apparatus, in which the procollagen granules containing cross-striations were often found, were remarkable elements in the IDE cells. During the stage of mineralisation, the IDE cells were tall columnar, and infoldings of distal plasma membrane of the IDE cells became marked. The most developed Golgi apparatus was visible at this stage, and large secretory granules containing fine granular or tubular materials were found in the distal cytoplasm that was close to the infoldings of the distal end. Many lysosomes that were ACPase positive were seen near the Golgi apparatus and in the distal cytoplasm of the IDE cells. ACPase positive granules often contained the cross-striation structure resembling procollagen, suggesting that the procollagen is degenerated in the IDE cells. During the maturation stage, the distal infoldings became unclear, and there were no large granules containing tubular materials, but many ACPase positive lysosomes were still present in the IDE cells. Non-specific ALPase was detected at the plasma membrane of the IDE cells at the mineralisation and maturation stages. K-NPPase was markedly detected at the plasma membrane of the IDE cells at the maturation stage. These results demonstrate that the IDE cells might be mainly involved in the removal of degenerated organic matrix from enameloid during the later formation stages. Strong Ca-ATPase activity was observed at the entire plasma membrane of the stratum intermedium cells, and there was slightly weak activity at the plasma membrane of the IDE cells during the mineralisation and maturation stages, implying that these cells are related to the active Ca transport to the maturing enameloid. It is likely that although the structure of the enamel organ is different, the function, especially at the mineralisation and maturation stages, is similar to other actinopterygians having well-mineralized cap enameloid.

This article reviews current findings on the organic matrix and the mineralization patterns in elasmobranchs, including an analysis of the role of the dental epithelial cells and the odontoblasts during odontogenesis. Our electron micrographs demonstrated that tubular vesicles limited by a unit membrane occupied the bulk of the elasmobranch enameloid matrix during the stage of enameloid matrix formation. It is likely that the tubular vesicles originated from the odontoblast processes. Two types of electron-dense fibrils, with cross-striations at intervals of approximately either 17 nm or 55 nm, respectively, were detected in the enameloid matrix. These data suggest that odontoblasts were strongly involved in enameloid matrix formation and in initial enameloid mineralization. Two types of odontoblasts, dark and light cells, were recognized during the stage of dentinogenesis. The light cells contained numerous mitochondria, intermediate filaments, and microtubules that extended their processes into the dentin. The darks cells possessed a well-developed Golgi apparatus and many cisternae in the rough endoplasmic reticulum, which suggests that the dark cells are involved in the formation of dentin. The inner dental epithelial (IDE) cells exhibited a well-developed Golgi apparatus, many mitochondria, cisternae of smooth endoplasmic reticulum, vesicles, vacuoles, and granules during the mineralization and maturation stages. During the stages of mineralization and early maturation, ACPase-positive granules were visible in the IDE cells and ALPase and Ca-ATPase activities were found at the lateral and proximal cell membrane of the IDE cells, suggesting that the IDE cells are involved in the removal of enameloid organic matrix and in the process of mineralization during later stages of enameloid formation. Our data indicate that elasmobranch enameloid is distinct from teleost enameloid, based on its organic content, on the mechanisms of its mineralization, and on the role of IDE cells concerning enameloid formation. Microsc. Res. Tech. 59:396–407, 2002. © 2002 Wiley-Liss, Inc.

The fine structure and the localization of nonspecific acid phosphatase (ACPase), nonspecific alkaline phosphatase (ALPase), and calcium-dependent adenosine triphosphatase (Ca-ATPase) activities in the dental epithelial cells in tooth germs of Dasyatis akajei in the later stages of enameloid formation were investigated. Numerous invaginations of the distal cell membrane of the inner dental epithelial (IDE) cells were observed at the early stage of enameloid maturation. The invaginations contain many fine granular and filamentous substances; the lamina densa, which was thicker during the former stages, is obscure. Granules exhibiting defined ACPase activity were usually found in the IDE cells during the stages of enameloid mineralization and maturation. IDE cells are putatively involved in the removal of degenerated enameloid matrix during these stages. Marked ALPase activity was detected at the proximal and the lateral cell membranes of the IDE cells from the late stage of enameloid matrix formation to the early stage of enameloid maturation. Strong activity of Ca-ATPase was localized at the proximal and the lateral cell membranes of the IDE cells during the stages of enameloid mineralization and maturation. ALPase and Ca-ATPase activity is probably related to crystal formation in the enameloid and the removal of degenerated enameloid matrix from the enameloid. J. Morphol. 252:170–182, 2002. © 2002 Wiley-Liss, Inc.

The structural features of the dental epithelial cells and the enameloid in tooth germs of the Japanese Port Jackson shark, Heterodontus japonicus, in the stages of enameloid formation, were investigated by light and transmission electron microscopy. At the enameloid matrix-formation stage, tall columnar inner dental epithelial cells contained large numbers of glycogen particles. At the enameloid mineralization stage, when many sharply outlined crystals appeared throughout the enameloid, the inner dental epithelial cells exhibited well-developed Golgi apparatuses and many mitochondria in the proximal cytoplasm, and abundant vesicles and vacuoles in the distal cytoplasm. Marked interdigitations of the lateral membrane were visible in the inner dental epithelial cells. The outer dental epithelial cells contained many mitochondria, lysosomal bodies, vesicles and microtubules, and the capillaries usually approached the outer dental epithelial cells. At the enameloid maturation stage, large numbers of crystals occupied the enameloid, and most of the organic matrix had disappeared from the enameloid area after demineralization. The organelles in the inner and outer dental epithelial cells decreased in number, but there were still widely distributed Golgi apparatuses, abundant intermediate filaments and granules containing an electron-dense substance in the inner dental epithelial cells. It is probable that the dental epithelial cells are involved in the removal of organic matrix from the enameloid and in the process of mineralization at the later stages of enameloid formation, i.e., the mineralization and the maturation stages.

Morphological features of the cap enameloid and dental epithelial cells were investigated by light and transmission electron microscopy during the various stages of enameloid mineralisation and early maturation in the tilapia. The pattern of mineralisation along collagen fibrils in the enameloid differed from that in the dentine. Many matrix vesicles were found in the predentine and in the enameloid, suggesting that they may be involved in the initial mineralisation in both regions. Most of the organic matrix disappeared from the cap enameloid during mineralisation and maturation. The disappearance of the organic matrix could be divided into 2 stages. Initially a fine network-like matrix, which probably consisted of glycosaminoglycans and extended between collagen fibrils, began to disappear. At the same time, fine crystallites and electron-dense, fine granular material covered the collagen fibrils as mineralisation of the enameloid began. In the second stage, the maturation of the enameloid, the collagen fibrils degenerated completely and disappeared from the cap enameloid, being replaced by large numbers of large crystals. At the mineralisation stage, the numbers of lysosomal bodies tended to increase in the inner dental epithelial (IDE) cells, which contained a well developed Golgi apparatus and rough endoplasmic reticulum (rER). At the early stage of maturation, a ruffled border was noted at the distal ends of the IDE cells, which contained many mitochondria and lysosomal bodies, but less rER. These features suggest that the cells actively absorb the organic matrix, which includes collagen fibrils, in the cap enameloid. The outer dental epithelial (ODE) cells were translucent cells that contained well developed labyrinthine canalicular spaces from the onset of the mineralisation stage to the middle stage of maturation. The IDE and ODE cells were clearly involved in the mineralisation of the cap enameloid at the mineralisation and maturation stages.

Ultra-thin resin sections of tooth bearing parts from three species were examined by electron microscopy. During enameloid matrix formation, matrix vesicles (MV) and fine aggregations of crystal-like structures probably derived from the MV were found in the enameloid matrix. In enameloid, however, no additional mineralization occurred along the collagen fibrils until mineralization had begun initially at the junction between the enameloid and orthodentine areas. Advance of mineralization along collagen fibrils was inhibited in the enameloid area before initial mineralization despite the presence of MV. Many MV were observed at the initial mineralization site; they appeared in the predentine during odontogenesis after the initial mineralization and were still visible during the stage of basal dentine formation. The odontoblasts continued to produce MV from the early stage of enameloid matrix formation until the late stage of basal dentine formation. However, despite their long appearance, MV are probably involved directly with the mineralization only when the initial mineralization starts.

The tooth plates in the living Holocephalan, Chimaera phantasma were studied by various techniques. They consisted of osteodentin and hypermineralized pleromin (kosmin). The degree of mineralization on the latter was as much as in the enameloid of Chondrichthyes and Osteichthyes. Scanning electron microscope observation indicated that the pleromin consisted of large and granular crystals. X-ray powder diffractometry, electron microprobe analysis and analytical electron microscopy revealed that the inorganic constituent of pleromin included, as an essential element, a beta-Ca3(PO4)2 (whitlockite) structure containing a small amount of Mg.